Ligand specificity in human glucose transporters GLUT1-5 and GLUT9

葡萄糖转运蛋白家族（GLUT、SLC 2）的成员参与糖尿病、肥胖症、癌症和其他疾病。 疾病有14种人GLUT，其在组织分布和底物亲和力和特异性方面不同。 由于缺乏特异性抑制剂和不充分的抗肿瘤药物， 了解它们运输差异的分子基础。几种GLUT的晶体结构和 它们的同源物非常相似，揭示了两种主要的转运蛋白构象，与一种 运输的交替机制。然而，底物亲和力和特异性的分子基础尚不清楚。还有， 迫切需要GLUT特异性配体，其可以进一步优化为药物、诊断标记物、 和调查工具，以探索特定GLUT在疾病中的作用。我们的长期目标是确定 碳水化合物转运的调节如何参与健康和疾病。在这方面的总体目标 本发明的应用是鉴定新的GLUT特异性配体和六种GLUT的配体特异性的分子基础： GLUT 1 -5和9。我们的中心假设是，GLUT中的配体特异性和亲和力取决于 转运蛋白的构象，并介导的底物结合位点残基和远程 涉及可溶性环的相互作用。我们关于GLUT 5和GLUT同系物Hxt、Gal 2和GLUT的初步数据显示， GlcPSe支持这一假设。拟议研究的基本原理是， 配体和负责配体识别和亲和力的GLUT特异性分子相互作用将使 开发GLUT的特异性调节剂，并最终实现其药理学控制。我们将测试我们的 具有以下两个特定目的的假设：1）鉴定GLUT 1 -5的构象特异性配体， GLUT 9;和2）确定GLUT 1 -5中负责配体特异性和亲和力的关键残基和区域 GLUT9对于第一个目标，我们将联合收割机结合小分子文库的计算机筛选， 和GLUT 1 -5和9模型的外向构象，体外验证了排名靠前的候选物， 抑制和选择性，在两个不同的GLUT个性化的运输系统。我们成立了 通过鉴定第一个有效和特异性的GLUT 5抑制剂来验证这种方法的可行性。第二个目标 突变体在GLUT特异性配体结合位点（已知的或由Aim测定的配体）中的转运活性 1)以及通过在所选的GLUT对之间交换可溶性环而构建的GLUT嵌合体，将被 检查底物和抑制剂特异性和/或亲和力的变化。配体结合位点将是 通过计算配体对接和配体GLUT的晶体结构测定确定 配合物该方法是创新的，因为它解决了GLUT家族中的配体特异性 通过组装方法的集成组合来全面地进行。拟议的研究意义重大， 因为它有望导致发现GLUT 1 -5和9的新型抑制剂和替代底物， 阐明了GLUT家族内功能变异的分子决定因素。

Members of the glucose transporter family (GLUT, SLC2) are involved in diabetes, obesity, cancer, and other diseases. There are 14 human GLUTs, which vary in tissue distribution and substrate affinity and specificity. Their exploitation as therapeutic targets has lagged due to lack of specific inhibitors and inadequate understanding of the molecular basis for their transport differences. Crystal structures of several GLUTs and their homologues are quite similar and reveal two major transporter conformations, consistent with an alternating mechanism of transport. Yet, the molecular basis of substrate affinity and specificity is unclear. Also, there is a critical need for GLUT-specific ligands that could be further optimized into drugs, diagnostic markers and investigative tools, to probe the role of particular GLUTs in disease. Our long-term goal is to determine how modulation of carbohydrate transport is involved in health and disease. The overall objective in this application is to identify new GLUT-specific ligands and the molecular basis of ligand specificity for six GLUTs: GLUT1-5 and 9. Our central hypothesis is that ligand specificity and affinity in GLUTs depends on the conformation of the transporter and is mediated by both substrate binding site residues and long-range interactions involving the soluble loops. Our preliminary data on GLUT5 and GLUT homologues Hxt, Gal2 and GlcPSe, support this hypothesis. The rationale for the proposed research is that identification of GLUT-selective ligands and GLUT-specific molecular interactions responsible for ligand recognition and affinity will enable development of specific modulators for GLUTs, and ultimately their pharmacological control. We will test our hypothesis with the following two specific aims: 1) identify conformation-specific ligands for GLUT1-5 and GLUT9; and 2) determine key residues and regions responsible for ligand specificity and affinity in GLUT1-5 and GLUT9. For the first aim, we will combine in silico screening of small molecule libraries against inward- and outward-facing conformations of GLUT1-5 and 9 models with in vitro validation of top ranked candidates, for inhibition and selectivity, in two different GLUT-individualized transport systems. We established the feasibility of this approach by identifying the first potent and specific inhibitor for GLUT5. For the second aim the transport activity of mutants in GLUT-specific ligand binding sites (ligands known or determined from Aim 1) and of GLUT chimeras constructed by swapping soluble loops between selected pairs of GLUTs, will be examined for changes in substrate and inhibitor specificity and/or affinity. Ligand binding sites will be determined through computational ligand docking and crystal structure determination of liganded GLUT complexes. The approach is innovative because it addresses ligand specificity in GLUT family comprehensively by assembling an integrated combination of methods. The proposed research is significant, because it is expected to lead to discovery of novel inhibitors and alternate substrates for GLUT1-5 and 9, and illuminate the molecular determinants of the functional variability within the GLUT family.